CN117551179A - 一种非洲猪瘟病毒多表位纳米颗粒疫苗 - Google Patents
一种非洲猪瘟病毒多表位纳米颗粒疫苗 Download PDFInfo
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Abstract
本申请属于动物疫苗制备技术领域,具体涉及一种非洲猪瘟病毒多表位纳米颗粒疫苗。该颗粒疫苗包含两种功能蛋白组分:SC‑Ferritin蛋白和ST‑XME蛋白;SC‑Ferritin蛋白氨基酸序列如SEQ ID No.1所示;ST‑XME蛋白氨基酸序列如SEQ ID No.4所示。本申请中,通过对特异性的B细胞表位和T细胞表位的筛选、识别,设计构建了相关免疫用蛋白。进一步利用铁蛋白作为载体以及将抗原与XCL1融合,靶向cDC1增强交叉提呈及T细胞的免疫应答,最终构建一种自组装纳米载体疫苗。初步结果表明,本申请的纳米颗粒疫苗能够以较低的抗原剂量来诱导高水平且持续的特异性抗体和特异性细胞免疫反应。
Description
技术领域
本申请属动物疫苗制备技术领域,具体涉及一种非洲猪瘟病毒多表位纳米颗粒疫苗。
背景技术
已有研究表明,非洲猪瘟病毒(ASFV)的p72、p30和CD2v蛋白均能够诱导猪体内产生中和抗体。其中:p72是ASFV的主要结构蛋白,占ASFV病毒颗粒的31%~33%,也是ASFV的主要靶标抗原之一;已有研究表明,pB602L作为p72的分子伴侣蛋白,属于一种非结构蛋白,缺乏pB602L会导致“拉链状”结构异常,病毒颗粒无法组装正常的二十面体形状,相关研究表明,pB602L和p30均具有高度抗原性,均可用于开发非洲猪瘟(ASF)疫苗和诊断工具;CD2v是ASFV的重要保护性抗原之一,可提供血清型特异性交叉保护免疫,并且CD2v是参与细胞活化、协同调控T细胞粘附分子CD2的唯一已知病毒同源物,因此,CD2v表位也是一种可用于构建ASF亚单位疫苗的重要靶点。
近年来,纳米疫苗因能携带抗原物质靶向递送至免疫器官,并且可以通过自我组装等方式制备而受到越来越多的关注。与传统疫苗相比,纳米颗粒更容易被抗原呈递细胞(Antigen presenting cell,APC)摄取,并且可以降低抗原的降解,提高抗原利用率,从而提高疫苗的作用效果。
树突状细胞(Dendritic cell,DC)在抗原呈递中发挥重要作用,被认为是体内专职APC(Antigen-presenting cells,抗原呈递细胞),在诱导和调节抗原特异性T细胞和B细胞中发挥关键作用。DC摄取并处理抗原,其表面会表达主要组织相容性复合体(Majorhistocompatibility complex,MHC),通过呈递抗原的MHC I类和II类分子与T细胞结合从而诱导适应性免疫的激活。趋化因子(Cmotif)受体1(XCR1)是DC的保守标记,XCR1也是唯一已知的XCL1受体。XCL1通过g蛋白偶联受体XCR1发挥作用,具有交叉呈递cDC1和激活CD8 T细胞,靶向XCR1可以提高DC的摄取效率,从而增强疫苗的保护效果。
总之,基于疫苗原理研究深入和新的疫苗设计理念的更新,针对非洲猪瘟病毒开发新的疫苗产品,对于确保非洲猪瘟病毒防治具有十分重要的技术意义。
发明内容
基于对B细胞和T细胞特定表位识别,本申请目的在于提供一种用于非洲猪瘟病毒防控的多表位纳米颗粒疫苗,从而为非洲猪瘟的防控奠定一定基础。
本申请所采取的技术方案详述如下。
一种非洲猪瘟病毒多表位肽,其序列如SEQ ID No.3所示。
利用所述非洲猪瘟病毒多表位肽所制备的ST-XME重组蛋白,其氨基酸序列如SEQID No.4所示;
对应的,ST-XME重组蛋白的编码核苷酸序列如SEQ ID No.5所示。
一种非洲猪瘟病毒多表位纳米颗粒疫苗,其包含两种功能蛋白组分:SC-Ferritin蛋白和ST-XME蛋白;
组装疫苗时,按SC-铁蛋白:ST-XME重组蛋白=24:1的摩尔比例,将SC-铁蛋白(SC-Ferritin)和ST-XME重组蛋白在缓冲液中(PBS缓冲液体系,pH值7.4)4℃孵育过夜;
所述ST-XME蛋白,其氨基酸序列如SEQ ID No.4所示;
所述SC-Ferritin蛋白,其氨基酸序列如SEQ ID No.1所示;
对应的,SC-Ferritin重组蛋白的编码核苷酸序列如SEQ ID No.2所示。
所述非洲猪瘟病毒多表位纳米颗粒疫苗的制备方法,具体包括如下步骤:
(一)制备重组载体
针对SC-Ferritin蛋白:
首先,按照前述SC-Ferritin重组蛋白的编码核苷酸序列(SEQ ID No.2),人工合成相关基因序列;随后,以pET-28a(+)质粒为载体,参考现有基因工程技术常规操作,将上述SC-Ferritin重组蛋白的编码核苷酸序列克隆重组到pET-28a(+)质粒中(BamHI/XhoI位点),构建获得重组质粒:SpyCatcher-Ferritin(SC-Ferritin);
针对ST-XME重组蛋白:
首先,按照前述ST-XME重组蛋白的编码核苷酸序列(SEQ ID No.5),人工合成相关基因序列;随后,以pET-28a(+)质粒为载体,参考现有基因工程技术常规操作,将上述ST-XME重组蛋白的编码核苷酸序列克隆重组到pET-28a(+)质粒中(BamHI/XhoI位点),构建获得重组质粒:SpyTag-XCR1-multi-epitope(ST-XME);
(二)转化、诱导蛋白表达
将步骤(一)中构建所得重组质粒SC-Ferritin、ST-XME分别转化已事先转化有pTf16伴侣质粒的大肠杆菌DE3感受态细胞,并筛选、鉴定获得正确的转化菌株;
将转化正确的菌株接种至含有双抗性的LB培养液(有氯霉素20μg/mL,卡那霉素50μg/mL)中,培养至OD600=0.6左右时,加入终浓度0.5mM的IPTG(异丙基-β-D-硫代半乳糖苷)和终浓度1.5mg/mL的阿拉伯糖,继续16℃培养12h(以诱导目的蛋白表达);
(三)蛋白纯化和纳米颗粒疫苗NanoFvax的组装(组合)
取步骤(二)中诱导表达结束后培养液,提取并纯化获得目的蛋白;
最后,按SC-铁蛋白:ST-XME重组蛋白=24:1的摩尔比例,将SC-铁蛋白(SC-Ferritin)和ST-XME重组蛋白在缓冲液中(PBS缓冲液体系,pH值7.4)4℃孵育过夜,以组装获得成品疫苗。
所述非洲猪瘟病毒多表位纳米颗粒疫苗在制备防治非洲猪瘟疫苗中的应用,应用时,以MF59和CpG-1826作为免疫佐剂。
基于表位位点来开发疫苗是一种较为常用的疫苗开发思路。本申请中,通过对特异性的B细胞表位和T细胞表位的筛选、识别,设计构建了相关免疫用蛋白。进一步地,为确保疫苗的有效递呈,发明人利用铁蛋白作为载体以及将抗原与XCL1融合,靶向cDC1增强交叉提呈及T细胞的免疫应答,来最终构建一种自组装纳米载体疫苗(铁蛋白是一种具有独特的笼状空间结构的载体蛋白,其可自组装成高度有序的24亚基聚合物,从而形成多个表面以展示抗原表位,最终提高抗原表位免疫原性和疫苗效力,并可将异质性降至最低)。初步实验结果表明,本申请所提供的纳米颗粒疫苗能够以较低的抗原剂量来诱导高水平且持续的特异性抗体和特异性细胞免疫反应。基于这些结果,本申请可为ASFV疫苗的开发或进一步完善奠定良好的技术基础。
附图说明
图1为不同抗原在B细胞表位的生物信息学分析(基于DNAStar Protean程序)预测结果;其中A、B、C、D分别为ASFV pB602L、p30、p72和CD2v抗原的表位分析预测结果;
图2为相关表位鉴定实验结果,其中:A为针对筛选所得B细胞表位的Dot blot实验结果,图中数字编号与表1中序号一致;B为针对T细胞表位的鉴定实验(基于免疫小鼠的特异性T细胞免疫反)结果;实验时,BALB/c小鼠分别肌肉注射重组蛋白CD2v或p72(30μg/只),免疫2周后,用ELISpot法检测多肽池刺激脾细胞后IFN-γ分泌情况,图中数字编号与表2中序号一致;
图3为筛选鉴定所得表位的保守性分析结果;其中中A、B、C、D分别为基于pB602L、p30、p72和CD2v鉴定筛选所得表位的保守性分析结果;彩色视图下,相同颜色表示氨基酸残基完全匹配,黑色虚线框表示已鉴定蛋白质表位的同源区域;
图4为筛选鉴定所得表位肽的三维模拟结构特征(使用Phyre2服务器预测)结果;其中A、B、C、D分别为基于pB602L、p30、p72和CD2v的模拟蛋白结果;所有图像均以表面模式显示,彩色视图下,表位区域以蓝色、红色、绿色和橙色显示;
图5为利用SpyTag-SpyCatcher连接酶系统制备多表位纳米颗粒疫苗的相关试验结果;其中:A为相关蛋白结果示意图;图中,SC:SpyCatcher,ST:SpyTag;Targeting unit指XCL1相关部分;B为纳米颗粒疫苗三维模拟示意图;彩色视图下,洋红色代表靶向分子,蓝色代表串联多表位,绿色代表铁蛋白;C为对制备所得蛋白的Western blot检测结果;图中泳道1为SC-铁蛋白,泳道2为ST-SME重组蛋白,泳道3为蛋白组合后纳米颗粒疫苗NanoFVax;图中M为蛋白Marker;D为对SC-铁蛋白(左)、纳米颗粒疫苗NanoFVax(右)的TEM观察结果以及粒径统计结果;E为基于DLS对SC-铁蛋白(左)、纳米颗粒疫苗NanoFVax的粒度分布统计结果;
图6为NanoFVax疫苗在体外诱导BMDC成熟及细胞因子变化情况的检测结果;其中:A、B、C分别为基于CD80+、CD86+和MHC-II+指标的流式检测结果;D、E分别为采用ELISA法对BMDC上清液中Th1细胞因子IL-2和Th2细胞因子IL-10的检测结果;F为对IL-21、CD40L和趋化因子CXCL10、CCL5的检测结果;
图7为NanoFVax疫苗蛋白的细胞摄取和体内生物分布分析结果;其为利用FITC标记的纳米颗粒疫苗NanoFVax处理DC细胞24h后的细胞摄取可视化结果;彩色视图中,细胞核为DAPI染色,细胞骨架用鬼笔环肽标记肌动蛋白,游离FITC用作对照;
图8为纳米颗粒疫苗注射小鼠后对小鼠免疫水平评价及特异性IgG、抗体亚型及细胞因子检测结果;其中:A为免疫实验及采样时间流程示意图;B为对不同时间采集所得血清样品中IgG抗体滴度测定结果;C为IgG亚型鉴定结果;D为对42d小鼠血清样品中IgG1/IgG2a比率测定结果;E为实验过程中小鼠体重变化统计结果;F为14d血清样品中各种细胞因子检测结果;
图8为纳米颗粒疫苗注射小鼠后对小鼠免疫水平评价及特异性IgG、抗体亚型及细胞因子检测结果;其中:A为免疫实验及采样时间流程示意图;B为对不同时间采集所得血清样品中IgG抗体滴度测定结果;C为IgG亚型鉴定结果;D为对42d小鼠血清样品中IgG1/IgG2a比率测定结果;E为实验过程中小鼠体重变化统计结果;F为14d血清样品中各种细胞因子检测结果;
图9为注射NanoFVax疫苗后对TFH细胞反应和T-/B细胞影响情况检测结果;其中:A为对TFH细胞(CD278+CD4+、CXCR5+PD-1+)的流式检测(右图)及对应统计结果(左图);B为对浆细胞(CD44+CD138+)的流式检测(右图)及对应统计结果(左图);C为对GC-B细胞(CD19+CD95+、GL7+CD95+、IgD+CD3+)的流式检测(右图)及对应统计结果(左图);D为对记忆B细胞(IgD+CD27+)的流式检测(右图)及对应统计结果(左图);
图10为注射NanoFVax疫苗后对体内细胞免疫反应影响情况;其中:A为CD4+细胞的流式检测(右图)及对应统计结果(左图);B为CD4+细胞的流式检测(右图)及对应统计结果(左图);C为对ELISpot刺激后产生的IFN-γ检测实验(右图)和统计结果(左图);D为对ELISpot刺激后产生的IL-4检测实验(右图)和统计结果(左图);E为利用MTT试验对淋巴细胞增殖情况检测统计结果;
上述各图及数据统计中,*p<0.05,**p<0.01,***p<0.001,****p<0.0001。
具体实施方式
下面结合实施例对本申请做进一步的解释说明。在介绍具体实施例前,就下述实施例中部分实验背景情况简要介绍说明如下。
生物材料:
大肠杆菌BL21(DE3)菌株,购自北京擎科生物技术股份有限公司;pTf16伴侣质粒,为现有技术中常用和常见质粒,可由公开渠道获得,申请人作为专业性教学和研究机构,因此,长期保存有相关质粒载体;
实验试剂:
IPTG、4%多聚甲醛、阿拉伯糖、DAPI,LPS、Triton X-100、青霉素/链霉素RPMI-1640培养基、MTT检测试剂盒、红细胞裂解液等,均为索莱宝公司产品;FBS(Fetal BovineSerum,胎牛血清),Gibco公司产品;IL-4、GM-CSF,BiosPacific公司产品;Ni-NTA柱,英国GEHealthcare公司产品;流式检测用相关抗体,均为美国BioLegend公司产品;鬼笔环肽-iFluor594,百奥莱博公司产品;HRP标记山羊抗小鼠IgG(H+L)IgG1、IgG2a、IgG2b、IgG2c和IgG3抗体,购自武汉三鹰公司;小鼠IL-2、IFN-γ、TNF-α、IL-12ELISA检测试剂盒,Invitrogen公司产品;小鼠IL-10、CD40L酶联免疫吸附试剂盒,购自于睿信生物技术公司;IFN-γ、IL-4ELIspot试剂盒,购自达科为公司;刀豆蛋白,美国Sigma-Aldrich公司产品;
主要仪器设备:
动态光散射仪,美国Wyatt Technology公司产品;透射电子显微镜,美国FEI公司产品;LSM 800共聚焦显微镜,德国蔡司公司产品;
小动物活体AMI成像系统,美国Spectral Instruments Imaging公司产品;流式细胞仪,BD公司产品;荧光酶联免疫斑点检测用CTL-S6分析仪,美国CTL公司产品。
实施例1
基于疫苗设计原理和前期工作基础,发明人以ASFV具有高抗原性的p72、p30、CD2v等蛋白为“抗原靶点”,对其在B细胞和T细胞中的免疫表位位点识别情况进行了分析,相关情况简介如下。
(一)相关病毒蛋白的优势B细胞表位
基于现有GenBank参考序列(GenBank:MK333180.1)和相关分析网站及工具,发明人对ASFV的pB602L、p30、p72和CD2v的优势B细胞表位进行了预测分析。
相关结果如图1及下表1所示。
表1,基于ASFV的pB602L、p30、p72和CD2v蛋白预测的B细胞表位
随后,参考上述预测结果人工合成相关多肽产物(委托金斯瑞生物科技股份有限公司合成提供)。
最后,利用Dot blot实验对所合成的多肽产物与抗ASFV血清的反应情况(评估ASFV阳性血清与合成的表位肽的亲和力)来对前述预测的B细胞表位进行筛选和验证。实验操作过程参考如下:取合成的多肽2μg(事先稀释)于NC膜上,用5%的BSA在37℃条件下封闭1h后,用PBST洗3次;再与抗ASFV血清孵育1h(37℃条件下);孵育结束后,对NC膜洗涤,加入HRP标记的鼠抗猪IgG二抗,再次孵育1h(37℃条件下);随后洗涤膜后,使用增强化学发光(ECL)显色底物以对蛋白质进行显影。
部分实验鉴定结果如图2所示。最终鉴定结果表明,B细胞中具有与ASFV阳性血清结合能力的表位肽序列共9条,具体如下:
针对pB602L蛋白,共3条:pB602L 39FKNDSRVAF47,pB602L 87TTKTLLSEL95,
pB602L 101TLKQETNDVPSES113;
针对p30蛋白,共2条:p30 16KTDLRSSSQV25,p30 75TEHQAQEEWNMI86,针对p72蛋白,共2条:p72 81TGTPTLGNKLTFGIP95,p72 279HFPENSHNIQTA290,针对CD2v蛋白,共2条:CD2v 34NDNNDINGVSWNF46,CD2v 84IFPHNDVFDTTYQ96。
(二)相关病毒蛋白的T细胞表位
利用现有分析网站和软件,发明人对ASFV的CD2v和p72蛋白在T细胞表面潜在结合位点(CD8 T细胞表位)进行了筛选(主要筛选预测与I类SLAs(swine leukocyte antigen)分子结合的多肽),其主要原理是:将与SLA-I分子结合的非肽映射到SLA-1*0401等位基因上,预测选出结合亲和力较高(共识分<0.5)和预测结合亲和力中等(共识分=0.5~1.0)的新肽。具体筛选预测结果如下表2所示。
表2,基于ASFV的CD2v和p72预测的SLA-1*0401等位基因的T细胞表位
随后,参考上述预测筛选结果人工合成相关多肽产物,并利用可视化小鼠IFN-γELIspot试剂盒进行ELISpot检测(多肽的刺激量为10μg/孔,相关操作参考其说明书进行即可),分析筛选预测的表位肽的特异性T细胞反应,从而确定可以激活T细胞的多肽序列。
验证结果如图2所示。最终ELIspot检测分析结果表明:针对p72的522ISDISPVTY530、和针对CD2v的150YTNESILEY158两个T细胞表位肽能被CD8+T细胞识别。
(三)多肽结构分析和蛋白结构模拟
利用现有分析工具,以ASFV Pig/HLJ/2018毒株为参考株,将现有不同基因型ASFV的p72、CD2v、pB602L和p30的蛋白质序列与ASFV/HLJ/2018毒株所鉴定出的表位蛋白质序列进行保守性比较(高度保守的优势表位可为确保ASFV疫苗的高免疫原性、广谱性保护和抗逃逸突变奠定基础),以分析验证前述筛选所得多肽序列的保守性情况。
部分结果如图3所示。分析结果表明:前述筛选所得表位序列在不同ASFV毒株中均高度保守,这可为相关疫苗制备奠定良好的基础。
进一步地,发明人使用Phyre2(http://www.sbg.bio.ic.ac.uk/phyre2)建立了蛋白三维模型(通过PyMol图形系统进生成图像),并分析了表位在蛋白三维结构的呈现特征。结果如图4所示。分析结果表明:p72、CD2v、pB602L和p30蛋白的其中九个表位位于蛋白表面,而CD2v 34NDNNDINGVSWNF46和pB602L 87TTKTLLSEL95的极个别氨基酸残基位于蛋白的内部。
总体而言,上述结果表明筛选所得的优势表位,可为ASFV高效疫苗开发奠定良好基础。
实施例2
基于纳米颗粒疫苗设计原理和发明人前期工作基础,本实施例中,发明人以铁蛋白作为ASFV多表位抗原递送载体,结合实施例1中基于生物信息学技术挖掘的抗原靶点,同时结合SpyTag/SpyCatcher系统,设计了一种纳米颗粒疫苗:NanoFvax。具体蛋白结构示意图如图5所示,即:
纳米颗粒疫苗NanoFvax包含两种功能蛋白组分:SC-Ferritin蛋白和ST-XME蛋白;
其中SC-Ferritin蛋白为Ferritin蛋白(铁蛋白)连接SpyCatcher后的重组蛋白(构建时,将铁蛋白序列借助于GSGESG连接子连接到SpyCatcher的C端,借助于基因工程技术手段,最终的重组蛋白以含有8×His-tag的融合蛋白形式表达);
ST-XME蛋白为重组免疫蛋白(重组免疫蛋白结构中,SpyTag位于XCL1的N端,依次连接B细胞和T细胞表位后,借助于基因工程技术手段,最终以8×His-tag的融合蛋白形式表达;其中每个免疫显性表位序列重复串联3次后使用柔性连接子GGGS连接;以pET28a(+)质粒为表达载体重组表达时,将免疫表位序列整体插入pET28a(+)质粒的BamHI和XhoI位点之间)。
SC-Ferritin蛋白相关编码基因序列及蛋白具体如下:
SpyCatcher(GenBank:MF974388.1),其氨基酸序列具体如下(139AA):MSYYHHHHHHDYDIPTTENLYFQGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDE DGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNE QGQVTVNGEATKGDAHT;
对应的编码核苷酸序列具体参考如下(417bp):
ATGTCGTACTACCATCACCATCACCATCACGATTACGACATCCCAACGACCGAAAACCTG
TATTTTCAGGGCGCCATGGTAACCACCTTATCAGGTTTATCAGGTGAGCAAGGTCCGTCC
GGTGATATGACAACTGAAGAAGATAGTGCTACCCATATTAAATTCTCAAAACGTGATGAG
GACGGCCGTGAGTTAGCTGGTGCAACTATGGAGTTGCGTGATTCATCTGGTAAAACTATT
AGTACATGGATTTCAGATGGACATGTGAAGGATTTCTACCTGTATCCAGGAAAATATACAT
TTGTCGAAACCGCAGCACCAGACGGTTATGAGGTAGCAACTGCTATTACCTTTACAGTTAATGAGCAAGGTCAGGTTACTGTAAATGGCGAAGCAACTAAAGGTGACGCTCATACT;Ferritin蛋白(铁蛋白,GenBank:WP_000949190.1),其氨基酸序列具体如下(167AA):MLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFL NENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYV AEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS;
对应的编码核苷酸序列具体参考如下(501bp):
ATGCTGAGCAAGGACATCATCAAGCTGCTGAACGAGCAGGTGAACAAGGAGATGAACA
GCAGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACACCCACAGCCTGGACGGCGC
CGGCCTGTTCCTGTTCGACCACGCCGCCGAGGAGTACGAGCACGCCAAGAAGCTGATC
ATCTTCCTGAACGAGAACAACGTGCCCGTGCAGCTGACCAGCATCAGCGCCCCCGAGC
ACAAGTTCGAGGGCCTGACCCAGATCTTCCAGAAGGCCTACGAGCACGAGCAGCACAT
CAGCGAGAGCATCAACAACATCGTGGACCACGCCATCAAGAGCAAGGACCACGCCACC
TTCAACTTCCTGCAGTGGTACGTGGCCGAGCAGCACGAGGAGGAGGTGCTGTTCAAGG
ACATCCTGGACAAGATCGAGCTGATCGGCAACGAGAACCACGGCCTGTACCTGGCCGACCAGTACGTGAAGGGCATCGCCAAGAGCAGGAAGAGC;
对应的,结合GSGESG连接子后,SC-Ferritin重组蛋白的氨基酸序列如SEQ IDNo.1所示;具体为(312AA):
MSYYHHHHHHDYDIPTTENLYFQGAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDE
DGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNE
QGQVTVNGEATKGDAHTGSGESGMLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSL
DGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISE
SINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS;
对应的,SC-Ferritin重组蛋白的编码核苷酸序列如SEQ ID No.2所示;具体为(936bp):ATGAGCTACTACCACCACCACCACCACCACGACTACGACATCCCCACCACCGAGAACCTGTACTTCCAGGGCGCCATGGTGACCACCCTGAGCGGCCTGAGCGGCGAGCAGGGCCCCAGCGGCGACATGACCACCGAGGAGGACAGCGCCACCCACATCAAGTTCAGCAAGCGCGACGAGGACGGCCGCGAGCTGGCCGGCGCCACCATGGAGCTGCGCGACAGCAGCGGCAAGACCATCAGCACCTGGATCAGCGACGGCCACGTGAAGGACTTCTACCTGTACCCCGGCAAGTACACCTTCGTGGAGACCGCCGCCCCCGACGGCTACGAGGTGGCCACCGCCATCACCTTCACCGTGAACGAGCAGGGCCAGGTGACCGTGAACGGCGAGGCCACCAAGGGCGACGCCCACACCGGCAGCGGCGAGAGCGGCATGCTGAGCAAGGACATCATCAAGCTGCTGAACGAGCAGGTGAACAAGGAGATGAACAGCAGCAACCTGTACATGAGCATGAGCAGCTGGTGCTACACCCACAGCCTGGACGGCGCCGGCCTGTTCCTGTTCGACCACGCCGCCGAGGAGTACGAGCACGCCAAGAAGCTGATCATCTTCCTGAACGAGAACAACGTGCCCGTGCAGCTGACCAGCATCAGCGCCCCCGAGCACAAGTTCGAGGGCCTGACCCAGATCTTCCAGAAGGCCTACGAGCACGAGCAGCACATCAGCGAGAGCATCAACAACATCGTGGACCACGCCATCAAGAGCAAGGACCACGCCACCTTCAACTTCCTGCAGTGGTACGTGGCCGAGCAGCACGAGGAGGAGGTGCTGTTCAAGGACATCCTGGACAAGATCGAGCTGATCGGCAACGAGAACCACGGCCTGTACCTGGCCGACCAGTACGTGAAGGGCATCGCCAAGAGCCGCAAGAGC。
ST-XME蛋白相关编码基因及蛋白序列如下:
SpyTag(GenBank:MF974389.1),其氨基酸序列(39AA)具体为:MGSSHHHHHHSSGLVPRGSVPTIVMVDAYKRYKGSGESG;
对应的编码核苷酸序列(117bp)具体为:
ATGGGCAGCAGCCACCACCACCACCACCACAGCAGCGGCCTGGTGCCCCGCGGCAGCGTGCCCACCATCGTGATGGTGGACGCCTACAAGCGCTACAAGGGCAGCGGCGAGAGCGGC;
XCL1(GenBank:NP_032536.1),氨基酸序列(114AA)具体为:
MRLLLLTFLGVCCLTPWVVEGVGTEVLEESSCVNLQTQRLPVQKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWVKAAIKTVDGRASTRKNMAETVPTGAQRSTSTAITLTG;
对应的编码核苷酸序列(GenBank:NM_008510.3,345bp)具体为:
ATGAGACTTCTCCTCCTGACTTTCCTGGGAGTCTGCTGCCTCACCCCATGGGTTGTGGAAGGTGTGGGGACTGAAGTCCTAGAAGAGAGTAGCTGTGTGAACTTACAAACCCAGCGGCTGCCAGTTCAAAAAATCAAGACCTATATCATCTGGGAGGGGGCCATGAGAGCTGTAATTTTTGTCACCAAACGAGGACTAAAAATTTGTGCTGATCCAGAAGCCAAATGGGTGAAAGCAGCGATCAAGACTGTGGATGGCAGGGCCAGTACCAGAAAGAACATGGCTGAAACTGTTCCCACAGGAGCCCAGAGGTCCACCAGCACAGCGATAACCCTGACTGGGTAA;
需要解释说明的是,前期相关研究中,还获得了三条免疫优势B细胞表位,具体如下:
针对pB602L蛋白:474SKENLTPDE482;针对p72蛋白:221MTGYKH;针对CD2v蛋白:160WNNSNINNFT169;
因此,利用柔性连接子连接后的具体免疫表位多肽序列如SEQ ID No.3所示,具体如下(624AA):
FKNDSRVAFGGGSFKNDSRVAFGGGSFKNDSRVAFIGGGSTTKTLLSELGGGSTTKTLLSELGGGSTTKTLLSELGGGSTLKQETNDVPSESGGGSTLKQETNDVPSESGGGSTLKQETNDVP
SESGGGSSKENLTPDEGGGSSKENLTPDEGGGSSKENLTPDEGGGSKTDLRSSSQVGGGSKT
DLRSSSQVGGGSKTDLRSSSQVGGGSTEHQAQEEWNMIGGGSTEHQAQEEWNMIGGGSTE
HQAQEEWNMIGGGSTGTPTLGNKLTFGIPGGGSTGTPTLGNKLTFGIPGGGSTGTPTLGNKL
TFGIPGGGSHFPENSHNIQTAGGGSHFPENSHNIQTAGGGSHFPENSHNIQTAGGGSMTGYK
HGGGSMTGYKHGGGSGGGSMTGYKHGGGSNDNNDINGVSWNFGGGSNDNNDINGVSW
NFGGGSNDNNDINGVSWNFGGGSIFPHNDVFDTTYQGGGSIFPHNDVFDTTYQGGGSIFPH
NDVFDTTYQGGGSWNNSNINNFTGGGSGGGSWNNSNINNFTGGGSWNNSNINNFTGGGSI
SDISPVTYGGGSISDISPVTYGGGSISDISPVTYGGGSYTNESILEYGGGSYTNESILEYGGGSYTNESILEYGGGS;
对应的,利用连接子连接后的ST-XME重组蛋白的氨基酸序列如SEQ ID No.4所示,具体如下(787AA):
MGSSHHHHHHSSGLVPRGSVPTIVMVDAYKRYKGSGESGGSGESGMRLLLLTFLGVCCLTP
WVVEGVGTEVLEESSCVNLQTQRLPVQKIKTYIIWEGAMRAVIFVTKRGLKICADPEAKWV
KAAIKTVDGRASTRKNMAETVPTGAQRSTSTAITLTGGGGSFKNDSRVAFGGGSFKNDSRV
AFGGGSFKNDSRVAFIGGGSTTKTLLSELGGGSTTKTLLSELGGGSTTKTLLSELGGGSTLK
QETNDVPSESGGGSTLKQETNDVPSESGGGSTLKQETNDVPSESGGGSSKENLTPDEGGGSS
KENLTPDEGGGSSKENLTPDEGGGSKTDLRSSSQVGGGSKTDLRSSSQVGGGSKTDLRSSS
QVGGGSTEHQAQEEWNMIGGGSTEHQAQEEWNMIGGGSTEHQAQEEWNMIGGGSTGTPT
LGNKLTFGIPGGGSTGTPTLGNKLTFGIPGGGSTGTPTLGNKLTFGIPGGGSHFPENSHNIQTA
GGGSHFPENSHNIQTAGGGSHFPENSHNIQTAGGGSMTGYKHGGGSMTGYKHGGGSGGGS
MTGYKHGGGSNDNNDINGVSWNFGGGSNDNNDINGVSWNFGGGSNDNNDINGVSWNFG
GGSIFPHNDVFDTTYQGGGSIFPHNDVFDTTYQGGGSIFPHNDVFDTTYQGGGSWNNSNIN
NFTGGGSGGGSWNNSNINNFTGGGSWNNSNINNFTGGGSISDISPVTYGGGSISDISPVTYGGGSISDISPVTYGGGSYTNESILEYGGGSYTNESILEYGGGSYTNESILEYGGGS;
对应的,ST-XME重组蛋白的编码核苷酸序列如SEQ ID No.5所示,具体如下(2361bp):ATGGGCAGCAGCCATCATCATCATCATCATAGCAGCGGCCTGGTGCCGCGCGGCAGCGTGCCGACCATTGTGATGGTGGATGCGTATAAACGCTATAAAGGCAGCGGCGAAAGCGGCGGCAGCGGCGAAAGCGGCATGCGCCTGCTGCTGCTGACCTTTCTGGGCGTGTGCTGCCTGACCCCGTGGGTGGTGGAAGGCGTGGGCACCGAAGTGCTGGAAGAAAGCAGCTGCGTGAACCTGCAGACCCAGCGCCTGCCGGTGCAGAAAATTAAAACCTATATTATTTGGGAAGGCGCGATGCGCGCGGTGATTTTTGTGACCAAACGCGGCCTGAAAATTTGCGCGGATCCGGAAGCGAAATGGGTGAAAGCGGCGATTAAAACCGTGGATGGCCGCGCGAGCACCCGCAAAAACATGGCGGAAACCGTGCCGACCGGCGCGCAGCGCAGCACCAGCACCGCGATTACCCTGACCGGCGGCGGCGGCAGCTTTAAAAACGATAGCCGCGTGGCGTTTGGCGGCGGCAGCTTTAAAAACGATAGCCGCGTGGCGTTTGGCGGCGGCAGCTTTAAAAACGATAGCCGCGTGGCGTTTATTGGCGGCGGCAGCACCACCAAAACCCTGCTGAGCGAACTGGGCGGCGGCAGCACCACCAAAACCCTGCTGAGCGAACTGGGCGGCGGCAGCACCACCAAAACCCTGCTGAGCGAACTGGGCGGCGGCAGCACCCTGAAACAGGAAACCAACGATGTGCCGAGCGAAAGCGGCGGCGGCAGCACCCTGAAACAGGAAACCAACGATGTGCCGAGCGAAAGCGGCGGCGGCAGCACCCTGAAACAGGAAACCAACGATGTGCCGAGCGAAAGCGGCGGCGGCAGCAGCAAAGAAAACCTGACCCCGGATGAAGGCGGCGGCAGCAGCAAAGAAAACCTGACCCCGGATGAAGGCGGCGGCAGCAGCAAAGAAAACCTGACCCCGGATGAAGGCGGCGGCAGCAAAACCGATCTGCGCAGCAGCAGCCAGGTGGGCGGCGGCAGCAAAACCGATCTGCGCAGCAGCAGCCAGGTGGGCGGCGGCAGCAAAACCGATCTGCGCAGCAGCAGCCAGGTGGGCGGCGGCAGCACCGAACATCAGGCGCAGGAAGAATGGAACATGATTGGCGGCGGCAGCACCGAACATCAGGCGCAGGAAGAATGGAACATGATTGGCGGCGGCAGCACCGAACATCAGGCGCAGGAAGAATGGAACATGATTGGCGGCGGCAGCACCGGCACCCCGACCCTGGGCAACAAACTGACCTTTGGCATTCCGGGCGGCGGCAGCACCGGCACCCCGACCCTGGGCAACAAACTGACCTTTGGCATTCCGGGCGGCGGCAGCACCGGCACCCCGACCCTGGGCAACAAACTGACCTTTGGCATTCCGGGCGGCGGCAGCCATTTTCCGGAAAACAGCCATAACATTCAGACCGCGGGCGGCGGCAGCCATTTTCCGGAAAACAGCCATAACATTCAGACCGCGGGCGGCGGCAGCCATTTTCCGGAAAACAGCCATAACATTCAGACCGCGGGCGGCGGCAGCATGACCGGCTATAAACATGGCGGCGGCAGCATGACCGGCTATAAACATGGCGGCGGCAGCGGCGGCGGCAGCATGACCGGCTATAAACATGGCGGCGGCAGCAACGATAACAACGATATTAACGGCGTGAGCTGGAACTTTGGCGGCGGCAGCAACGATAACAACGATATTAACGGCGTGAGCTGGAACTTTGGCGGCGGCAGCAACGATAACAACGATATTAACGGCGTGAGCTGGAACTTTGGCGGCGGCAGCATTTTTCCGCATAACGATGTGTTTGATACCACCTATCAGGGCGGCGGCAGCATTTTTCCGCATAACGATGTGTTTGATACCACCTATCAGGGCGGCGGCAGCATTTTTCCGCATAACGATGTGTTTGATACCACCTATCAGGGCGGCGGCAGCTGGAACAACAGCAACATTAACAACTTTACCGGCGGCGGCAGCGGCGGCGGCAGCTGGAACAACAGCAACATTAACAACTTTACCGGCGGCGGCAGCTGGAACAACAGCAACATTAACAACTTTACCGGCGGCGGCAGCATTAGCGATATTAGCCCGGTGACCTATGGCGGCGGCAGCATTAGCGATATTAGCCCGGTGACCTATGGCGGCGGCAGCATTAGCGATATTAGCCCGGTGACCTATGGCGGCGGCAGCTATACCAACGAAAGCATTCTGGAATATGGCGGCGGCAGCTATACCAACGAAAGCATTCTGGAATATGGCGGCGGCAGCTATACCAACGAAAGCATTCTGGAATATGGCGGCGGCAGC。
基于基因工程技术,纳米颗粒疫苗NanoFvax的制备方法具体介绍说明如下。
(一)制备重组载体
针对SC-Ferritin蛋白:
首先,按照前述SC-Ferritin重组蛋白的编码核苷酸序列(SEQ ID No.2),人工合成相关基因序列;随后,以pET-28a(+)质粒为载体,参考现有基因工程技术常规操作,将上述SC-Ferritin重组蛋白的编码核苷酸序列克隆重组到pET-28a(+)质粒中(BamHI/XhoI位点),构建获得重组质粒:SpyCatcher-Ferritin(SC-Ferritin);
针对ST-XME重组蛋白:
首先,按照前述ST-XME重组蛋白的编码核苷酸序列(SEQ ID No.5),人工合成相关基因序列;随后,以pET-28a(+)质粒为载体,参考现有基因工程技术常规操作,将上述ST-XME重组蛋白的编码核苷酸序列克隆重组到pET-28a(+)质粒中(BamHI/XhoI位点),构建获得重组质粒:SpyTag-XCR1-multi-epitope(ST-XME);
(二)转化、诱导蛋白表达
将步骤(一)中构建所得重组质粒SC-Ferritin、ST-XME分别转化已事先转化有pTf16伴侣质粒的大肠杆菌DE3感受态细胞(采用该伴侣质粒以增加蛋白质翻译表达后的溶解度);具体转化操作可参考如下:
冰上融化事先转化有pTf16伴侣质粒的大肠杆菌DE3感受态细胞后,加2μL(1ng/mL)步骤(一)中构建所得重组质粒,轻轻混匀后,冰上放置30min;
随后,将上述混合物置于42℃水浴中热激30s,立即置于冰上并加入450μL培养液(LB培养液),混合均匀后,37℃、225rpm摇床震荡培养1h;
再后,吸取100μL菌液均匀涂布双抗性LB平板(有氯霉素20μg/mL,卡那霉素50μg/mL)上,37℃、倒置培养过夜;
最后,挑取阳性克隆进一步进行菌液PCR鉴定以及测序鉴定,确保转化正确。
挑选上述转化正确的菌株,进一步转接至LB培养液(有氯霉素20μg/mL,卡那霉素50μg/mL)中,培养至OD600=0.6左右时,加入终浓度0.5mM的IPTG(异丙基-β-D-硫代半乳糖苷)和终浓度1.5mg/mL的阿拉伯糖,继续16℃培养12h(诱导目的蛋白表达)。
诱导蛋白表达过程中、以及结束后,分别取样进行免疫印迹试验分析检测。结果表明(分结果如图5所示),SC-铁蛋白(SC-Ferritin)和ST-XME重组蛋白分子量分别约为35kDa和65kDa,这一结果与预期相符合,也初步表明成功制备获得了相关目的蛋白。
(三)蛋白纯化和纳米颗粒疫苗NanoFvax的组装(组合)
取步骤(二)中诱导表达结束后培养液,提取并纯化获得目的蛋白,具体操作参考如下:取1L培养液进行离心(9000rpm、15min),收集菌体沉淀,PBS清洗菌体沉淀后,加入PBS重悬后,冰浴、超声破碎80min以裂解菌体;
随后,对上述菌体破碎后裂解液进行离心(4℃、15000r/min离心1h),收集上清液,并对上清利用Ni-Sepharose 6Fast Flow树脂(GE Healthcare)进行纯化。具体纯化操作可参考如下:将上清液上样加至Ni-NTA亲和层析柱中,样品全部加完后,分别使用20mM、200mM咪唑洗脱并最终获得纯化蛋白;其中,20mM咪唑使用30个柱体积洗脱,其余浓度咪唑均洗脱10个柱体积。
对所得纯化蛋白分别进行SDS-PAGE检测分析,以确保所得均为目的蛋白。
将纯化所得SC-铁蛋白(SC-Ferritin)和ST-XME重组蛋白进一步组合(组装)成为纳米颗粒疫苗NanoFvax时,具体操作如下:
按SC-铁蛋白:ST-XME重组蛋白=24:1的摩尔比例,将前述所得纯化后SC-铁蛋白(SC-Ferritin)和ST-XME重组蛋白在缓冲液中(PBS缓冲液体系,pH7.4)4℃孵育过夜,以组装成为纳米颗粒疫苗NanoFvax(组装时,铁蛋白和表位肽可以形成分子间的异肽键,从而将铁蛋白与抗原亚基结合为一个整体,示意图如图5所示)。
对组装后纳米颗粒疫苗NanoFvax进行蛋白免疫印迹实验检测,结果表明(如图5所示),终产物纳米颗粒疫苗的分子量与两种蛋白(SC-铁蛋白、ST-XME单体)的分子量之和结果一致,表明两种蛋白成功连接为一个整体(通过spvtag/spycatcher连接)。
采用负染色方法,进一步对组装后的纳米颗粒疫苗形态进行透射电子显微镜(TEM)观察(观察操作时:将样品在PBS中稀释至终浓度为0.1mg/mL后,取5μL样品悬浮液滴加在铜网上,静置数分钟,然后用滤纸去除多余液体,滴入醋酸铀酰溶液1min,干燥后进行电子显微镜观察(图像由Tecnai G2 spirit BioTwin(FEI)拍摄)。结果如图5所示。可以看出:TEM结果显示铁蛋白和组装成纳米颗粒后的NanoFVax均呈球形结果,这一结果表明抗原表位肽可以成功的负载于铁蛋白其表面,并且不会影响铁蛋白后续被进一步作为抗原进行呈递。
进一步采用动态光散射法(DLS),对铁蛋白及组装后的纳米颗粒尺寸进行表征。结果如图5所示。分析结果表明:在PBS溶液中,NanoFVax纳米粒子的Z平均直径为86.10nm,铁蛋白的平均直径约为20nm。
从上述TEM和DLS数据结果可以看出,组装后所制备的纳米颗粒疫苗NanoFvax样品大小均匀、分散良好。
实施例3
针对实施例2所制备的纳米颗粒疫苗,发明人进一步进行了体外细胞实验和初步的动物实验,具体情况简介如下。
(一)DC细胞对纳米颗粒疫苗的响应情况以BALB/c小鼠(6-10周龄)的骨髓树突状细胞(BMDC)为实验材料,对DC细胞对纳米颗粒的反应情况进行了实验分析。具体过程及结果简介如下。
(1)分离获得BMDC
采用颈椎脱臼法处死小鼠,无菌条件下取出股骨和胫骨,剪去骨两端后,用PBS冲洗出骨髓至培养皿中;利用70μm的细胞筛网,对所收集的骨髓悬液进行过滤(去除小碎片和肌肉组织);对滤液进行离心,1200rpm离心5min,弃上清;加入红细胞裂解液,重悬细胞,室温孵育5min后,200rpm离心5min,弃上清;PBS洗1次,然后用含10% FBS的RPMI-1640培养液重悬细胞,至此已获得小鼠骨髓细胞;将该细胞在含有10% FBS和1%青霉素/链霉素的RPMI-1640培养基中培养,同时添加IL-4(10ng/mL)和GM-CSF(20ng/mL),37℃、5%CO2条件下培养至第7d,收获未成熟的BMDC并立即使用。
(2)刺激BMDC
取将上述步骤(1)中所得未成熟的BMDC,加入到96孔板中(1×105个/孔),用LPS(100ng/mL)、单体(ST-XME重组蛋白,30μg/mL)和NanoFvax(30μg/mL)处理48h;
处理结束后,取样测定IL-2和IL-10细胞因子分泌情况来评估Th1和Th2的极化反应情况(测定时,取处理后培养上清,利用小鼠IL-2酶联免疫吸附试剂盒和小鼠IL-10酶联免疫吸附试剂盒,参考其说明书进行操作检测即可);
同时,对LPS、单体和NanoFvax处理后细胞,用0.25%(v/v)胰蛋白酶-EDTA消化分离并离心处理后,将细胞转移到新的实验容器中,PBS缓冲液重新悬浮后,采用流式细胞术评估DC成熟标志物情况(使用FITC标记的抗小鼠CD80、PE标记的抗小鼠CD86和抗小鼠MHC-II,检测这些标志物在BMDC细胞表面的表达情况)。
IL-2和IL-10在调节T细胞增殖和功能方面具有重要作用。基于ELISA方法利用相关试剂盒对BMDC上清液中细胞因子IL-2和IL-10水平的检测结果如图6所示(图6D、图6E)所示。可以看出:经NanoFVax处理后,BMDC中IL-2(平均130pg/mL)和IL-10(平均150pg/mL)的水平相较于PBS组(IL-2 78pg/mL,IL-10 34pg/mL)显著升高;而由于这些细胞因子分别由Th1和Th2细胞分泌,也即,这一结果表明NanoFVax可同时促进Th1和Th2的细胞因子分泌。
流式细胞术对BMDC的成熟和抗原呈递能力的检测结果如图6所示(以CD80、CD86和MHC II为检测指标,以100ng/mL的LPS处理作阳性对照)。分析可以看出:NanoFVax处理48h后的BMDCs,由图6A、图6B可知,CD80+(53.5%)和CD86+(58.3%)细胞的百分比明显高于单体组的CD80+(44.7%)和CD86+(41.3%);同时,图6C显示纳米颗粒疫苗处理还增加了BMDCs中MHC II的表达(60.6%),结果表明经过NanoFVax处理后有效刺激了BMDC的成熟,并使其抗原呈递能力得到了增强。
进一步地,发明人对成熟BMDC上清液中IL-21、CD40L以及趋化因子CXCL10和CCL5的分泌情况进行了检测分析。结果如图6F所示。分析可以看出:
NanoFVax处理后BMDC诱导了较高水平的细胞因子与趋化因子的分泌;与单体刺激的IL-21(平均2pg/mL)、CD40L(平均0.3pg/mL)、CXCL10(平均6pg/mL)和CCL5(平均5pg/mL)的分泌量,以及PBS刺激的IL-21(平均7pg/mL)、CD40L(平均2.4pg/mL)、CXCL10(平均110pg/mL)和CCL5(平均25pg/mL)的分泌量相比,NanoFvax刺激组的IL-21(平均19pg/mL)、CD40L(平均4.5pg/mL)、CXCL10(平均210pg/mL)和CCL5(平均57pg/mL)的分泌量显著升高。
上述结果均表明,NanoFVax不仅能刺激BMDC成熟,还能增强BMDC的抗原呈递,而这些都是诱导T细胞免疫反应以及激活免疫细胞的重要前提。
(3)DC细胞摄取纳米颗粒疫苗后胞内分布情况
对所制备的纳米颗粒疫苗进行NHS-FITC标记(利用FITC偶联试剂盒,参考其说明书进行操作)后,采用共聚焦荧光成像技术对DC细胞摄取纳米颗粒疫苗后疫苗在胞内分布情况进行了观察分析。具体操作过程参考如下:在24孔板中低密度培养DC2.4细胞过夜后,加入FITC标记的NanoFVax(20μg/孔)孵育24h;弃细胞培养上清液,PBS轻洗细胞,细胞用4%多聚甲醛室温固定30min;对固定后细胞用0.1%的PBS冲洗后,用0.1% Triton X-100/PBS通透10min,再使用鬼笔环肽-iFluor 594(1:1000)染色F-肌动蛋白,使用DAPI染细胞核;最后,利用共聚焦显微镜(Zeiss LSM800)进行观察和对荧光强度进行统计分析。
DC作为体内专职的APC在诱导保护性免疫中发挥着关键作用。抗原内化是后续DC活化和抗原交叉呈递的重要前提。通过对疫苗进行荧光标记进而利用显微镜观察是直观判定疫苗能否被宿主免疫系统识别和处理的直观技术手段。相关结果如图7所示。分析可以看出:荧光信号显著增强,绿色荧光主要分布在细胞胞质中,占比36.56%,相较于MOCK组的1.25%具有显著差异。这一结果表明,NanoFVax可被DC快速摄取并内化,纳米颗粒可被DC有效吞噬,从而更有利于抗原的呈递。
实施例4
上述实验基础上,针对实施例2所制备纳米颗粒疫苗,发明人进行了进一步的动物免疫实验,相关实验过程及结果简介如下。
(一)动物免疫
将雌性BALB/c小鼠(7周龄左右)经7d适应性饲喂后,进行分组(每组5只):实验组,在第0d、14d和28d在大腿处肌肉分别注射单体和纳米颗粒疫苗NanoFVax(以MF59和CpG-1826用作免疫佐剂(注射时具体用量,质量比计,疫苗NanoFVax:MF59:CpG-1826=1:1:1);对照组,同样方式、同样时机注射同样用量的PBS;实验期间(流程如图8A所示),小鼠自由饮食和活动,定期观察和记录小鼠体重等情况,并定期从小鼠尾静脉采集血清样本以备检测(样本采集后立即-80℃保存备检)。
(二)相关检测项目及结果
实验过程中,通过对相关免疫指标检测分析结果,以综合评估本申请所制备的纳米颗粒疫苗的实际效果。具体检测项目及结果检测如下。
(1)抗体亚型及滴度
针对所采集的血清样本(分别于7、14、21、28d采集),采用ELISA法检测血清中的抗体滴度和亚型。具体操作过程参考如下:利用碳酸盐缓冲液(pH=9.6)将纯化的幽门螺杆菌铁蛋白(SC-Ferritin蛋白)进行稀释,然后按300ng/孔的量包被于96孔板上(4℃过夜);包被完成后,PBST清洗并拍甩多余液体后,使用5% BSA进行封闭(PBS稀释),37℃封闭处理1h;封闭完成后,用PBST冲洗,加入100μL倍比稀释的小鼠血清样品,37℃孵育1h;然后,分别加入HRP标记的山羊抗小鼠IgG(H+L)、IgG1、IgG2a、IgG2b、IgG2c及IgG3抗体,再次37℃孵育1h;孵育结束后,PBST洗3次,加入双组份TMB显色液,室温避光孵育15min后,加入2M H2SO4(50μL/孔)终止反应;最后在450nm波长处检测吸光度。
免疫过程中,对免疫后小鼠体重监测结果表明,与对照组相比,NanoFVax免疫组的小鼠体重基本不受影响(图8E所示)。这一结果表明本申请所提供的纳米颗粒疫苗没有明显的副作用。
血清样品特异性IgG滴度测定结果表明:NanoFvax免疫组产生高滴度特异性抗体,并在第4周(第3次接种时)诱导的特异性抗体滴度达到4.096×106,显著高于PBS组和单体组(平均抗体滴度1.254×104)(图8B);单体疫苗组虽然也能诱导抗原特异性抗体,但5周后抗体水平明显下降;而注射NanoFVax后的抗体滴度在19周内一直保持在较高水平,之后才出现轻微下降趋势(图8B);另外,对每只小鼠产生抗体的时间过程分析表明,注射纳米颗粒疫苗的BALB/c小鼠在注射第二周后出现高效价的特异性抗体(平均抗体滴度3.989×103)(图8B)。
总体上,纳米颗粒诱导的特异性抗体滴度明显高于其他组别,并且在231d时抗体滴度水平(1.28×105)仍处于较高水平。这一结果表明,NanoFVax显著增强了抗原的免疫原性,从抗体滴度和抗体持续时间来看,NanoFVax比单体疫苗能引起更强的体液免疫反应。
对免疫2周后血清样本的特异性IgG亚型检测结果表明:纳米颗粒NanoFvax组诱导的IgG1抗体水平略高,IgG2a、IgG2b和IgM水平相似;同时,IgG2c和IgA抗体水平在各组表现为较低水平(图8C所示)。
IgG1/IgG2a的比率通常被用作Th1/Th2介导反应的指标。此次实验中,NanoFVax免疫小鼠的特异性IgG1滴度高于特异性IgG2a滴度,IgG1/IgG2a比值大于1且大于其他组,表明NanoFVax诱导了更多的Th1和Th2免疫应答,并且表现出略有倾斜的抗原特异性Th2反应(图8D)。
(2)细胞因子水平检测
按照生产商的说明,使用ELISA试剂盒检测血清中的IL-2、IFN-γ、TNF-α、IL-12(Invitrogen,Waltham,MA,USA)和IL-4、IL-10(瑞新生物,南京,中国)。使用标准曲线计算细胞因子浓度。使用Spark 20M平板阅读器(TECAN,瑞士曼内多夫)分析细胞因子概况。
对免疫42天后血清中细胞因子水平测定结果表明:注射NanoFVax后,IL-2(70pg/mL)、IFN-γ(280pg/mL)、IL-4(140pg/mL)、TNF-α(125pg/mL)、IL-10(180pg/mL)和IL-12(75pg/mL)相较于单体组IL-2(18pg/mL)、IFN-γ(60pg/mL)、IL-4(45pg/mL)、TNF-α(85pg/mL)、IL-10(30pg/mL)和IL-12(40pg/mL)和PBS组显著增加。这一结果表明疫苗能引起标志性的细胞免疫反应,进一步表明体内激活的免疫细胞偏向于Ⅰ型细胞免疫反应,同时伴有Ⅱ型细胞免疫反应(图8F)。
(3)ELISPOT(固相酶联免疫斑点)检测
取最后一次免疫14d后小鼠,参考前述操作,采集脾脏组织并处理,最终将小鼠脾细胞在含10%FBS和青霉素/链霉素的RPMI-1640培养基中制备单细胞悬液;检测操作时,用无血清培养基将细胞稀释至2×106个/m后,取样加入96孔ELISpot板中(每孔100μL);加入纯化蛋白(10μg/孔),孵育36h;具体操作参考试剂盒(小鼠IFN-γ和IL-4ELIspot试剂盒)说明书即可;最后,使用CTL-S6分析仪对斑点进行成像,并进行定量分析;通过计算IFN-γ和IL-4阳性T细胞的数量来评估抗原特异性T细胞响应情况。
基于ELISpot法对脾细胞预处理上清液中细胞因子IFN-γ和IL-4分泌情况检测结果表明:NanoFVax预处理的PBMC所诱导分泌的IFN-γ(320/200万个脾细胞)和IL-4(540/200万个脾细胞)相比较于单体所诱导分泌的IFN-γ(205/200万个脾细胞)和IL-4(170/200万个脾细胞)显著提高(图10C、图10D)。
(4)流式细胞检测
参考前述说明,将免疫后小鼠脾脏单细胞悬液调整为2×105个/mL后,取样置于流式细胞术检测用试管中;根据检测项目,对细胞用萤光素标记的单克隆流式抗体进行孵育标记(4℃、避光孵育30min,使用的流式抗体包括:抗-CD3 APC、抗-CD4 FITC、抗-CD8 FITC、抗-CD19APC、抗-GL7 PE、抗-IgD PE、抗-CD20 FITC、抗-CD95 FITC、抗-PD-1APC、抗-CD44PE、抗-CD25 FITC,以及抗-CD69 APC、抗-CD278 FITC、抗-CXCR5 FITC、抗-CD138 FITC、抗-CD27 FITC);孵育结束后,对细胞使用PBS清洗,并重悬于1mL/管的PBS中,流式细胞仪检测分析。
利用流式细胞术对TFH细胞(T滤泡辅助细胞)(CD278+CD4+CXCR5+PD-1+)、浆细胞(CD44+CD138+)、GC B细胞(CD19+CD95+GL7+IgD+)和记忆B细胞(IgD+CD27+)的检测结果表明(图9):NanoFVax免疫组所诱导的TFH细胞(CD278+CD4+44.7%、CXCR5+PD-1+49.8%)、GC B细胞(CD44+CD138+47.8%)、GC B细胞(CD19+CD95+56.0%、CD95+GL7+59.3%)和记忆性B细胞(IgD+CD27+45.0%)数量相较于PBS免疫组所诱导的TFH细胞(CD278+CD4+3.78%、CXCR5+PD-1+25.2%)、GC B细胞(CD44+CD138+18.7%)、GC B细胞(CD19+CD95+20.3%、CD95+GL7+33.7%)和记忆性B细胞(IgD+CD27+25.2%)及单体免疫组所诱导的TFH细胞(CD278+CD4+27.9%、CXCR5+PD-1+27.3%)、GC B细胞(CD44+CD138+39.4%)、GC B细胞(CD19+CD95+34.1%、CD95+GL7+41.2%)和记忆性B细胞(IgD+CD27+35.3%)显著增高。这一结果表明,NanoFVax能够有效刺激体液免疫反应。
流式细胞术对三免后第二周的小鼠脾脏CD3+T细胞的增殖及其分化CD4+和CD8+T细胞的检测结果表明(图10A、图10B,T细胞反应在保护BALB/c小鼠免受ASFV感染中起着至关重要的作用):与PBS(6.62%)和单体抗原(19.9%)的免疫效果相比,NanoFVax免疫组激活的CD3+CD4+T细胞数量(25.2%)显著增加(p<0.001),NanoFVax诱导的CD8+T细胞数量(14.04%)高于单体抗原(7.09%)和PBS(5.9%)(p<0.05),NanoFVax诱导的细胞免疫偏向辅助性T细胞免疫,CD4+T细胞多于CD8+T细胞。
(5)淋巴细胞增殖
将免疫3次后的BALB/c小鼠安乐死后,无菌条件下分离脾脏组织并剪碎处理后(PBS缓冲液中处理),置于RPMI-1640培养基中,进一步研磨处理后过70μm规格细胞筛处理后,1200g离心5min,弃上清;在细胞沉淀中加入5mL红细胞裂解缓冲液重悬细胞后静置5min,1200g离心5min,弃上清,用RPMI-1640培养基洗涤1次,以去除已裂解的红细胞;
再后,将剩余的脾细胞重悬于含10%胎牛血清的RPMI-1640培养基中,并调整至1×106个/mL备检。
实验检测时,刺激指数测定时,参考如下操作过程:
将处理过的淋巴细胞接种到96孔板后,以纯化的纳米颗粒及单体抗原(5μg/mL)作为刺激剂;以ConA(10μg/mL)处理作为阳性对照;以RPMI-1640培养基作为阴性对照;置于37℃、5% CO2的培养箱中培养48h;培养结束后,向细胞中加入3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四氮唑噻唑蓝(MTT)溶液,再培养4h后,小心去除MTT溶液,加入DMSO溶解MTT甲氮;最后,在490nm波长下测量平均光密度,并根据实验组生长量与阴性组生长量之比计算刺激指数。
通过使用NanoFvax和单体两种抗原刺激物来评估小鼠淋巴细胞的增殖活性,结果显示(图10E):在阳性对照LPS的刺激下,所有实验组的淋巴细胞增殖能力都高于对照组(5.13%)。此外,NanoFvax组刺激的淋巴细胞增殖能力(14.8%)强于单体刺激的淋巴细胞增殖能力(7.6%)(p<0.05)。
总体上,本申请以ASFV蛋白对B细胞和T细胞免疫优势抗原表位为基础,结合针对cDC1上XCR1趋化因子受体,以铁蛋白作为纳米载体以及利用SpyTag/SpyCatcher蛋白连接酶系统来制备获得了一种米颗粒疫苗,其可以靶向DC从而提高抗原摄取和呈递效果,进而有效激发免疫反应效果。相关试验结果也表明,该纳米颗粒疫苗能促进DC吞噬和成熟,并有利于淋巴结富集,最终可产生持久的体液免疫反应,同时能显著诱导增强T细胞和B细胞免疫反应,可为ASFV疫苗开发奠定良好的技术基础,也为相关新疫苗开发提供了较好的借鉴和指导。
Claims (9)
1.一种非洲猪瘟病毒多表位肽,其特征在于,所述表位肽序列如SEQ ID No.3所示。
2.利用权利要求1所述非洲猪瘟病毒多表位肽所制备的ST-XME重组蛋白,其特征在于,所述ST-XME重组蛋白的氨基酸序列如SEQ ID No.4所示。
3.权利要求2所述ST-XME重组蛋白的编码基因,其特征在于,所述编码基因的核苷酸序列如SEQ ID No.5所示。
4.一种非洲猪瘟病毒多表位纳米颗粒疫苗,其特征在于,该颗粒疫苗包含两种功能蛋白组分:SC-Ferritin蛋白和ST-XME蛋白;
所述SC-Ferritin蛋白,其氨基酸序列如SEQ ID No.1所示;
所述ST-XME蛋白的氨基酸序列如SEQ ID No.4所示。
5.如权利要求4所述非洲猪瘟病毒多表位纳米颗粒疫苗,其特征在于,组装疫苗时,按SC-铁蛋白:ST-XME重组蛋白=24:1的摩尔比例,将SC-Ferritin蛋白和ST-XME蛋白在缓冲液中4℃孵育过夜。
6.权利要求4所述非洲猪瘟病毒多表位纳米颗粒疫苗的制备方法,其特征在于,具体包括如下步骤:
(一)制备重组载体
针对SC-Ferritin蛋白:
首先,按照SEQ ID No.2所示SC-Ferritin蛋白的编码核苷酸序列,制备对应的基因序列;
随后,以pET-28a(+)质粒为载体,将SC-Ferritin蛋白的编码核苷酸序列克隆重组到pET-28a(+)质粒中,构建获得重组质粒:SpyCatcher-Ferritin;
针对ST-XME重组蛋白:
首先,按照SEQ ID No.5所示ST-XME蛋白的编码核苷酸序列,制备对应基因序列;
随后,以pET-28a(+)质粒为载体,将ST-XME蛋白的编码核苷酸序列克隆重组到pET-28a(+)质粒中,构建获得重组质粒:SpyTag-XCR1-multi-epitope;
(二)转化、诱导蛋白表达
将步骤(一)中构建所得重组质粒分别转化大肠杆菌感受态细胞,并筛选、鉴定获得正确的转化菌株;
将转化正确的菌株培养扩增后,加入IPTG和阿拉伯糖诱导蛋白表达;
(三)蛋白纯化和纳米颗粒疫苗NanoFvax的组装(组合)
取步骤(二)中诱导表达结束后培养液,提取并纯化获得蛋白;
最后,将分别获得的SC-铁蛋白和ST-XME蛋白组装成疫苗。
7.权利要求1所述非洲猪瘟病毒多表位肽在制备防治非洲猪瘟疫苗中的应用。
8.权利要求4所述所述非洲猪瘟病毒多表位纳米颗粒疫苗在制备防治非洲猪瘟疫苗中的应用。
9.如权利要求8所述非洲猪瘟病毒多表位纳米颗粒疫苗在制备防治非洲猪瘟疫苗中的应用,其特征在于,应用时,以MF59和CpG-1826作为免疫佐剂。
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